Pharmaceutical compositions comprising tyrphostins

ABSTRACT

Compounds useful for countering undesired toxic effects to cells, tissues or organs having formula (I) wherein: Ar is a group of formulae (i) or (ii), n is O or, when Ar has formula (i) above, then n may also be 1, R is CN, —GC(S)NH2, —C(O)NHR3 or, when R1 is 4—NO2 and R2 is H or 3—OH, then R may also be a group of formulae (iii), (iv), (v), (vi) where R3 is H, phenyl, phenyl(lower alkyl) or pyridylmethyl; R1 and R2 are each independently H, OH, NO2 or, when R is CN, also CH3, F, or CF3, provided that both R1 and R2 are simultaneously H.

FIELD OF THE INVENTION

[0001] The present invention concerns compositions which are useful tocounter damage caused by harmful agents, particularly anti-neoplasticagents used in cancer treatment, e.g. cytotoxic drugs. Damage in thecontext of the present invention means adverse effects on either cells,tissue or organs. The present invention also concerns therapeuticmethods to counter such damage. Furthermore, the present inventionconcerns also novel compounds useful in such compositions and methods.

BACKGROUND OF THE INVENTION

[0002] Most of the commonly used antineoplastic treatments, includingchemotherapy and radiation, have adverse toxic effects, manifested ondividing cells and in the function of certain organs. Cells which areparticularly affected by such treatment are bone marrow cells, skincells and cells of the gastrointestinal tract epithelium. In addition,such treatments often cause damage to specific organs, such as thekidney and liver. As a result of such toxic effects, the therapeuticindex of such treatments is limited.

[0003] It is a long felt want in medical research to try and developtreatment modalities and means which will reduce the unwanted toxic sideeffects, without affecting the therapeutic activity of the drugs, thusincreasing their therapeutic index.

[0004] Apoptosis or programmed cell death is a fundamental physiologicalmechanism of cell death regulated during embryonal development andnormal homeostasis mechanisms within the body. Recent data indicatedthat the anti tumor effect of a variety of chemotherapeutic agents isrelated to their ability to induce apoptosis. The toxicity of theseagents may also be related to the induction of apoptosis in normalcells.

[0005] Preliminary reports have described use of various pharmacologicalagents for the prevention of nephrotoxicity resulting from the use ofanti-cancer drugs (Skinner, R., Current Opinion in Oncology, 7:310-315,1995). Attempts were made to counteract the severe chronic proximaltubular toxicity resulting from use of the cytotoxic drug Cisplatin. Inin vivo experiments in rats, para-aminobenzoic acid (PABA)co-administered together with cisplatin resulted in reducednephrotoxicity of the cisplatin without reducing its anti-tumoractivity. Other agents such as mitimazole, chloropromazin and L-argininewere also administered to animals in various in vivo experiments carriedout in connection with toxicity of cisplatin resulting in onlypreliminary non-conclusive findings.

[0006] The drug amifostine which is dephosphorylated to yield a thiolmoiety was also used for reducing anti-toxic effects of anti-neoplasticdrugs (Cancer Res., 55:4069, 1995).

GENERAL DESCRIPTION OF THE INVENTION

[0007] The present invention provides, by a first of its aspects, apharmaceutical composition for countering, i.e. reducing or preventing,damage to cells or tissue comprising, as an active agent, an effectiveamount of a compound of the general formula I:

[0008] wherein:

[0009] Ar is a group of the formulae

[0010] n is 0 or, when Ar has the formula (i) above, then n may also be1, R is CN, —C(S)NH₂, —C(O)NHR₃ or, when R₁ is 4-NO₂ and R₂ is H or3-OH, then R may also be a group of the formula

[0011] where R₃ is H, phenyl, phenyl(lower alkyl) or pyridylmethyl;

[0012] R₁ and R₂ are each independently H, OH, NO₂ or, when R is CN,also CH₃, F, or CF₃, provided that both R₁ and R₂ are not simultaneouslyH. together with a pharmaceutically acceptable carrier.

[0013] Said active agent and said pharmaceutical composition may beadministered to or contacted with cells or tissue in a variety ofconditions to reduce or prevent undesired damage to cells, tissues ororgans. Examples of such conditions are such which may lead toapoptosis. Preventing of undesired apoptosis is a specific embodiment ofthe invention. Such conditions may also be exposure of said cells,tissue or organ to harmful factors, which may be exogenous or endogenousfactors, as well as other physiological conditions, which may lead todamage, e.g. change in temperature, impairment of blood flow, exposurean ionizing irradiation, etc. The damage to be prevented may also be aresult of natural physiological deterioration processes, e.g. suchoccurring in cells, tissue or organs, maintained, grown or cultured exvivo.

[0014] The present invention also provides a method of treatment of anindividual, for countering damage to cells, tissue or organ, comprisingadministering to the individual an effective amount of a compound havingthe general formula I.

[0015] The term “effective amount” should be understood as meaning anamount of the active compound, which is effective in countering damagemanifested in either destruction of normal (non-diseased) cells ordamage to a tissue or organ.

[0016] The harmful factor may be an exogenous agent e.g., a therapeuticdrug having a cytotoxic effect (e.g. anti-neoplastic drugs),irradiation, noxious chemicals, etc. In addition, the harmful factor maybe endogenous, such as free radicals the level of which rises in thecourse of various metabolic or other disorders, auto-antibodies,cytokines, etc.

[0017] The pharmaceutical compositions of the invention may be used inthe framework of treatment of various diseases, disorders or conditionssuch as AIDS, conditions which may give rise to hepatotoxicity,radiation injuries, reducing or inhibiting damage to grafted cells ortissue as a result of graft rejection, for the treatment ofintoxications, e.g,. paracetamol intoxication, for countering adverseeffects of noxious solvents and carriers of therapeutic drugs,countering alcohol-caused damages, etc. Furthermore, the compositionsmay also be used for countering non-desired immune mediated reactions oran inflammatory response, e.g. septic shock, to reduce damage caused byautoimmune reactions and others. Finally, the pharmaceuticalcompositions of the invention also may be used in ex vivo preservationof cells, tissues or organs used for transplantation to reduce orprevent cell or tissue deterioration or death which may otherwise occurduring the time they are kept ex vivo prior to transplantation, etc.

[0018] The pharmaceutical compositions of the invention are especiallyuseful to counter toxic effects of cytotoxic or anti-metabolic drugs,particularly such as used in cancer therapy. Occasionally, thepharmaceutical composition of the invention will be administered inconjunction with the cytotoxic drug. A pharmaceutical compositionaccording to this embodiment may comprise such a drug in combinationwith a compound of above formula I.

[0019] The compounds of formula I belong to a family of compounds knownas Tyrphostin compounds (Levitzki, A. and Gazit, A. Science, 267:1782,1995). In the following text, a compound of Formula I will be referredto as “Tyrphostin” and compositions comprising such compounds will attimes be referred to as “Tyrphostin compositions”.

[0020] Out of the tyrphostins of formula I, some are known, albeit foruses other than those provided by the invention, and others are novel.The novel tyrphostins, which constitute another, independent aspect ofthe invention, are those of formula I, wherein Ar, R₁ R₂ and R₃ are asdefined above, with the provisos that

[0021] a) R cannot be

[0022] b) when R is CN and n is 0, then

[0023] (ba) if one of R₁ and R₂ is H or OH, then the other cannotrepresent NO₂;

[0024] (bb) if one of R₁ and R₂ is H or F, then the other cannotrepresent H or F; and

[0025] c) when R₁ is 4-NO₂, R₂ is H and n is 0, then R cannot represent—C(O)NH₂ or —C(S)NH₂.

[0026] Preferred compounds of formula I for use in the compositions arethose wherein R is CN, —C(S)NH₂, —C(O)NHCH₂C₆H₅ or a group of theformula

[0027] and n is 0, R₁ is 4-NO₂ and R₂ is H.

[0028] Examples of tyrphostins are shown in compound Table I. Some ofthe tyrphostins shown in Compound Table I are novel, and these noveltyrphostins are shown also in Compound Table II. Compound Table I

No. R R1 R2 m.p. ° C. Ref¹ AG1714 CN 4-NO₂ H 153 1 AG1791

4-NO₂ H 238 2 AG1744

4-NO₂ H 277 3 AG1820

4-NO₂ H 103 4 AG1822

4-NO₂ H 220 — AG1801

4-NO₂ H 181 — AG1824

4-NO₂ H 238 — AG1823

4-NO₂ H 187 — AG1798

4-NO₂ H 98 — AG1745

4-NO₂ H 148 — AG1843

4-NO₂ H — — AG126 CN 4-NO₂ 3-OH 176 5 AG1719

4-NO₂ 3-OH 215 — AG1720

4-NO₂ 3-OH 105 — AG1759 CN 4-NO₂ 3-CH₃ 97 — AG1762 CN 4-NO₂ 3-F 120 —AG1606

4-NO₂ 3-OH 148 — AG1821

4-NO₂ 3-OH 178 6 AG1781 CN 3-NO₂ H 90 10 AG127 CN 3-NO₂ 4-OH 175 11AG178 CN 4-F H 114 11, 12 AG1754 CN 3-F 4-F 57 13 AG1755 CN H 4-CF₃ 95 —AG1799 CN 4-NO₂

158 —

[0029] Compound Table II (Novel Compounds)

No. R R1 R2 m.p. ° C. AG1822

4-NO₂ H 220 AG1801

4-NO₂ H 161 AG1824

4-NO₂ H 238 AG1823

4-NO₂ H 187 AG1798

4-NO₂ H 98 AG1745

4-NO₂ H 148 AG1843

4-NO₂ H — AG1719

4-NO₂ 3-OH 215 AG1720

4-NO₂ 3-OH 105 AG1759 CN 4-NO₂ 3-CH₃ 97 AG1762 CN 4-NO₂ 3-F 120 AG1606

4-NO₂ 3-OH 148 AG1755 CN H 4-CF₃ 95 AG1799 CN 4-NO₂

158

List of References for Several Known Compounds Used in Accordance withthe Invention

[0030] 1. Mowry, D. T., J. Am. Chem. Soc., 67:1050, 1945

[0031] 2. Zabichy, J., J. Chem. Soc., 683, 1961.

[0032] 3. Bronskill, J. S., Dc A., and Ewing, D. F., J. Chem. Soc.Perkin, Trans. I., 629, 1978

[0033] 4. Junck, H., and Wolny, B., Monat. Chem., 107:999, 1976

[0034] 5. Novogrodsky, A.. Vanichkin, A., Patya, M., Gazit, A., Osherov,N. and Levitzki, A., Science, 264:1319, 1994

[0035] 6. Sakamoto, M., et al., J. Chem. Soc., Perkin Trans I., 1759,1995

[0036] 7. (a) Flenner, A. L., C. A. 63:13,278, 1965

[0037] (b) Lythgoe, K. Todd, A. and Tapham, C. J. Chem. Soc., 515, 1944

[0038] 8. Drabek, J. and Meyer, A., C. A., 89:152,719, 1978

[0039] 9. Gazit, A., et al., J. Med. Chem., 34:1896, 1991

[0040] 10. Carson, B. B., and Staughton, R. W., J. Am. Chem. Soc.,50:2825, 1928.

[0041] 11. Gazit, A., et al., J. Med. Chem., 32:2344, 1989

[0042] 12. Weinberger, M. A., and Higgin, R. M., Can. J. Chem., 43:2585,1965

[0043] 13. Blox Ram, J., Dell, C. P., and Smith, C. W., Heterocycles,38:400, 1994.

[0044] Particularly preferred tyrphostins in accordance with theinvention are the compounds designated as AG1714, AG1744, AG1801 andAG1843 in the above “Compound Table I”. Out of these four compounds, thelatter two are novel.

[0045] The tyrphostins of formula I may be administered to the patientin combination with anothcr treatment, e.g,. in combination with acytotoxic drug or irradiation. In such a combination treatment thetyrphostins may be administered simultaneously with or at a differenttime than the other treatment, so as to yield a maximum effect. Aparticular example is administration of the tyrphostins prior to theother treatment, e.g. several hours prior to the irradiation or to theadministration of the cytotoxic drug.

[0046] It was found in accordance with the invention that when thetyrphostins of formula I are administered together with anothertherapeutic agent, they do not reduce the therapeutic activity of theother agent, but rather act in reducing its undesired toxic side effectsto normal cells or tissue. This means that the therapy with thetherapeutic agent will still achieve the same desired therapeuticeffect, at the same dosage. For example, in the case of achemotherapeutic drug, the administration of a tyrphostin will not, ormay only minimally affect the effect of the drug in reducing the tumorload or preventing tumor growth or tumor recurrence, at a givenadministration dosage. In some cases, however, administration of thetyrphostins even intensifies the therapeutic effect of the treatment.The overall effect of the tyrphostins, in such a combination therapy, isthus the increase in the therapeutic index of another therapy, namely,increase in the ratio between the therapeutic effect of the therapy toits undesired toxic side effects. The increase in therapeutic index mayat times be used to advantage of increasing the dosage of thetherapeutic agent, e.g. the dosage of the cytotoxic agent or radiation,without a concurrent increase in undesired toxic side effects.

[0047] The tyrphostins according to the invention may be administeredeither in a single dose or may be given repetitively over a period oftime, e.g. prior, during and after the treatment with a cytotoxic agentor irradiation.

[0048] A preferred mode of administration of the tyrphostins to humansis intravenously, although by a proper formulation, they may also beadministered by other administration modes, e.g. intramuscularly,intraperitoneally or orally.

[0049] While the tyrphostin compositions will typically contain a singletyrphostin compound it is possible at times to include in thecomposition and or co-administer two or more tyrphostins which acttogether in a synergistic or additive manner to counter damages caused,e.g. by a therapeutic treatment.

BRIEF DESCRIPTION OF THE FIGURES

[0050]FIG. 1A is a graphic representation showing the mortality of micetreated with doxorubicin as compared to the mortality of mice receivinga single injection of AG1714 two hours before treatment withdoxorubicin.

[0051]FIG. 1B is a graphic representation showing, the mortality of micetreated with cisplatin as compared to the mortality of mice receiving asingle injection of AG1714 two hours before treatment with cisplatin.

[0052]FIG. 2 is a graphic representation showing the mortality of micereceiving 10 i.p. injections of doxorubicin (5 mg/kg) for a period of 21days (cumulative dose 50 mg/kg) as compared to the mortality of micetreated as above but receiving an i.p. injection of AG1714 (5 mg/kg) twohours prior to each doxorubicin injection.

[0053]FIG. 3 is a graphic representation showing the mortality of micetreated with cisplatin alone or with cisplatin and AG1801 over a periodof ten days after cisplatin administration.

[0054]FIG. 4 is a graphic representation showing the mortality of micetreated with doxorubicin alone or with tyrphostins and doxorubicin overa period of ten days after doxorubicin administration.

[0055]FIG. 5 is a graphic representation showing the effect of a numberof tyrphostins on the toxic effect of cisplatin in kidneys of treatedmice. The toxic effect is measured by the level of creatinine detectedin the serum of the treated mice, a high level of creatinine indicatingkidney damage (nephrotoxicity).

[0056]FIG. 6 is a graphic representation showing the level of creatinine(FIG. 6A) and blood urea nitrogcn (BUN) (FIG. 6B) (which are indicativeof nephrotoxicity) in serum of mice receiving either cisplatin alone,AG1714 alone or a combination of cisplatin and AG1714. The levels ofcreatinine and BUN were compared to the levels of the same substances inserums of control mice receiving the vehicle only.

[0057]FIG. 7 is a photograph showing histopathological analysis ofkidneys (FIG. 7A-C) and small intestines (FIG. 7D-F) of miceadministered with cisplatin alone or with the tyrphostin AG1714 prior tothe administration of cisplatin. Final magnification ×400;

[0058]FIG. 7A—kidneys of normal non-treated mice;

[0059]FIG. 7B—kidneys of mice receiving cisplatin alone showing largeproteinaceous plaques in the proximal tubules;

[0060]FIG. 7C—kidneys of mice receiving AG1714 prior to cisplatinadministration showing normal kidney structure;

[0061]FIG. 7D—small intestines of non-treated mice;

[0062]FIG. 7E—small intestine of mice receiving cisplatin alone showingsevere necrosis and damage;

[0063]FIG. 7F—a small intestine of mice treated with AG1714 prior tocisplatin administration showing normal small intestine structure;

[0064]FIG. 8 is a graphic representation showing the level of twotransaminases, Aspartic transaminasc (AST) and Alanine-transaminase(ALT) in serum of mice receiving an injection of anti-FAS antibody aloneor in combination with the tyrphostin AG1801. High levels of thetransaminases indicate anti-FAS antibody induced damage to the liver(hepatotoxicity);

[0065]FIG. 9 is a graphic representation showing the level of twotransaminases AST and ALT in serum of mice receiving an injection ofanti-FAS antibody alone or in combination with the tyrphostin AG1714;High levels of the transaminases indicate anti-FAS antibody induceddamage to the liver (hepatotoxicity);

[0066]FIG. 10 is a graphic representation showing the level of AST andALT in serum of mice treated with Con A alone or in combination withAG1714;

[0067]FIG. 11 is a graphic representation showing the level of AST andALT in serum of mice treated with TNF/GalN or in combination with thetyrphostin AG1714;

[0068]FIG. 12 is a graphic representation showing the number of bonemarrow nucleated cells (FIG. 12A) or the number of colony forming units(CFU) (FIG. 12B) in femur bone marrow of mice treated with doxorubicinalone, AG1714 along or with their combination;

[0069]FIG. 13 is a graphic representation showing the number ofnucleated cells in bone marrow of mice treated with doxorubicin alone orwith AG1714 and doxorubicin:

[0070]FIG. 13A shows the number of nucleated cells in bone marrow of themice receiving different doses of doxorobucin;

[0071]FIG. 13B shows the number of nucleated cells in bone marrow, ofthe treated mice at different periods of time after treatment withdoxorubicin;

[0072]FIG. 14 is a graphic representation showing the weight of thespleen (FIG. 14A) and thymus (FIG. 14B) of mice treated either withdoxorubicin alone or with AG1714 prior to doxorubicin administration;

[0073]FIG. 15 is a graphic representation showing the number of bonemarrow nucleated cells in the femurs of mice treated with 5FU alone orwith 5FU and AC1714;

[0074]FIG. 16 is a graphic representation showing the number of bonemarrow nucleated cells in the femurs of mice treated with mitomycin-Calone or with mitomycin-C and AG1714;

[0075]FIG. 17 is a graphic representation showing the number ofnucleated cells in bone marrow of mice treated with doxorubicin alone orwith AG1801 at different doses prior to doxorubicin administration;

[0076]FIG. 18 is a graphic representation showing the number ofnucleated cells in bone marrow of irradiated mice (300R) and of micetreated with the tryphostin AG1714 before irradiation;

[0077]FIG. 19 is a graphic representation showing the number of cells inbone marrow of irradiated mice (450R) and of mice treated withtyrphostin AG1714 before irradiation;

[0078]FIG. 20 is a graphic representation showing the mortality of miceafter lethal irradiation (800R) as compared to the mortality of micetreated with tyrphostin AG1714 prior to irradiation over a period of 23days after irradiation;

[0079]FIG. 21 is a graphic representation showing the weight of lungs ofmice inoculated with MCA-105 fibrosarcoma cells (FIG. 21A), Lewis lungcarcinoma cells (FIG. 21B) or B-16 melanoma cells (FIG. 21C) and treatedwith either cisplatin alone, AG1714 alone or with a combination ofcisplatin and AG1714;

[0080]FIG. 22 is a graphic representation showing the weight of nudemice bearing the SK-28 human melanoma tumors and treated withdoxorubicin, AG1714 or their combination;

[0081]FIG. 23 is a graphic representation showing the volume of thehuman ovary carcinoma tumor (OVCAR-3) in mice treated either withcisplatin alone, with AG1714 alone or with their combination;

[0082]FIG. 24 is a graphic representation showing the volume of theOVCAR-3 tumor in mice receiving repetitive administration of thetreatments described in FIG. 23 above;

[0083]FIG. 25 is a graphic representation showing the weight of lungs inMCA-105 fibrosarcoma tumor bearing mice treated with cisplatin alone,AG1801 alone or a combination of both (FIG. 25A) or with doxorubicinalone, AG1801 alone or their combination (FIG. 25B);

[0084]FIG. 26 is a graphic representation showing the weight of lungs ofmice treated with cisplatin alone, AG1801 alone or a combination ofboth;

[0085]FIG. 27 is a graphic representation showing the number of colonyforming units (CFU) in cultures of bone marrow cells grown with orwithout various concentrations of AG1714;

[0086]FIG. 28 is a graphic representation showing the viability ofthymocytes grown with or without AG1714 at various periods of time afterthe addition of AG1714 to the cells;

[0087]FIG. 29 is a graphic representation showing the number of beatsper minute in cardiomyocyte cultures grown with or without AG1714 as anindication of the viability of the cells;

[0088]FIG. 30 is a graphic representation showing the percent lysismeasured by percent of specific ⁵¹Cr release of target EL-4 cells bymurine peritoneal exudate cytotoxic lymphocytes (PEL) in the presence orabsence of AG1714.

PREPARATIVE EXAMPLES

[0089] The preparation of several of the above novel compounds isexemplified in the following preparative examples. In all the examplesbelow, the new compounds were synthesized by knoevenagel condensation ofthe aldehyde with malononitrile or the substituted amide.

Example 1—AG1801

[0090]0.45 g, 3 mM, p-nitro benzaldehyde, 0.61 g, 3.5 mM, N-(Cyanoacetyl) benzyl amide (reference 9) and 50 mg β-alanine in 20 ml ethanolwere refluxed for 5 hours. Cooling and filtering gave 0.67 g, 73% yield,light yellow solid, mp-164°. NMR(CDCl₃) δ 8.44 (1H,S,vinyl), 8.35, 8.07(4H,AB,J_(AB)=8.8 Hz), 7.35 (5H,m), 6.6(1H,br.t,NH), 4.63(2H,d,J=5.8Hz).

[0091] MS-m/e 307(M⁺, 50%), 290(M—HCN,63), 260(M—H—NO₂,91),201(M—NHCH₂C₆H₅,15), 173 (M—CONHCH₂C₆H₅,13), 155(22), 127(21), 105(100).

Example 2—AG1798

[0092] 0.4 g, 2.65 mM, p-nitrobenzaldehyde, 0.57 g, 2.8 mM, N(cyanoacetyl) 3-propyl phenyl amide (reference 9) and 40 mg β-alanine in 30 mlethanol were refluxed for 4 hours. The solution was concentrated byevaporation, cooled and filtered to give 0.38 g, 43% yield, light-yellowsolid, mp-98.

[0093] NMR (CDCl₃) δ 8.38(1H,S, vinyl), 8.35, 8.06(4H,ABq, J_(AB)=8.6Hz), 7.3(5H,m), 3.50(2H, J=8.0 Hz), 2.74(2H,t,J=8.0 Hz), 2.0(2H,quintet, J=8.0 Hz).

Example 3—AG1719

[0094]146 mg, 0.87 mM, 3-hydroxy 4-nitro benzaldehyde, 48 mg, 0.89 mM,malononitrile dimer and 20 mg β-alanine in 10 ml ethanol were refluxedfor 1 hour. Evaporation, trituration with CH₂Cl₂-hexane and filteringgave 190 mg, 78% yield, yellow solid, mp-105. NMR (acetone d₆) δ8.32(1H,d,J=8.6 Hz), 8.20(1H,S,vinyl), 7.77(1H,d,J=2.2 Hz),7.65(1H,dd,J=8.6, 2.2 Hz).

Example 4—AG1761

[0095] 170 mg, 1 mM, 3-fluoro 4-nitro benzaldehyde, 80 mg, 1.2 mM,malononitrile and 15 mg β-alanine were refluxed for 1 hour. Evaporation,trituration with hexane and filtering gave 202 mg, 93% yield, pinksolid, mp-120°. NMR (CDCl₃) δ 8.22(1H,m), 7.83(3H,m).

[0096] MS-m/e 217(M⁺,100), 187(M—NO,78), 171(M—NO₂,19),159(M—NO—HCN—H,80), 151(M—NO₂—HF,33), 144(M—NO₂—HCN,71), 132(75),124(M—NO₂—HCN—HF,81).

Example 5—AG1799

[0097] 0.4 g, 2.65 mM, 4-nitro benzaldehyde, 0.51 g, 2.8 mM, phenylsulphonyl acetonitrile and 40 mg β-alanine in 30 ml ethanol wererefluxed for 4 hours. Cooling, filtering and washing with ethanol gave0.68 g, 82% yield, light yellow solid, mp-158°. NMR (CDCl₃) δ8.36(2H,d,J=8.6 Hz), 8.31(1H,S, vinyl), 8.07(4H,m), 7.70(3H,m).

EXAMPLES OF BIOLOGICAL AND THERAPEUTIC ACTIVITIES

[0098] The biological and therapeutic effect of some of the compoundswhich may be used in the composition of the invention will now beexemplified in the following, non-limiting examples with occasionalreference to the annexed figures.

I. Reduction of Mouse Mortality Caused by Doxorubicin or Cisplatin byTyrphostins Example 6

[0099] 6 weeks old female CD1 mice were divided into the followinggroups:

[0100] i. Mice injected intraperitoneally (i.p.) with doxorubicin (20mg/kg);

[0101] ii. Mice injected i.p. with cisplatin (14 mg/kg);

[0102] iii. Mice receiving a single i.p. injection of AG1714 (20 mg/kg)two hours prior to the doxorubicin; and

[0103] iv. Mice receiving a single i.p. injection of AG1714 (20 mg/kg)two hours prior to receiving the cisplatin.

[0104] As seen in FIG. 1, treatment of mice with AG1714 significantlyreduced the mortality of the mice as compared to the mortality of micereceiving doxorubicin alone (p≦0.001) (FIG. 1A) or of mice receivingcisplatin alone (p≦0.005) (FIG. 1B).

Example 7

[0105] 6 weeks old female CD1 mice were divided into the following twogroups:

[0106] i. Mice receiving 10 i.p. injections of doxorubicin at aconcentration of 5 mg/kg per each injection over 21 days (cumulativedose of 50 mg/kg,); and

[0107] ii. Mice receiving 10 i.p. injections of doxorubicin at 5 mg/kgover 21 days with the addition of a single i.p. injection of AG1714 (5mg/kg) two hours prior to each doxorubicin injection.

[0108] Each group consisted of 10 mice and the % mortality in each groupwas tested as explained above.

[0109] As seen in FIG. 2, the mortality of the mice receiving injectionsof AG1714 prior to each administration of doxorubicin was significantlyreduced as compared to the high mortality of the mice receivingdoxorubicin treatment alone.

Example 8 Two groups of CD1 mice were injected with 14 mg/kg ofcisplatin as described above. One group received a single i.p. injectionof AG1801 at a low dose of 1 mg/kg, two hours before receiving thecisplatin injection. As seen in FIG. 2 all the mice in the groupreceiving cisplatin alone died within ten days of the injection. Againstthis, in the group of mice receiving a single injection of AG1801 at alow dose prior to the cisplatin injection, the % mortality was only 20%.Example 9

[0110] CD1 mice were divided into the following five groups:

[0111] i. Mice receiving a single i.p. injection of 20 mg/kg ofdoxorubicin;

[0112] ii. Mice receiving a single i.p. injection of 20 mg/kg of AG1714and two hours later an i.p. injection of 20 mg/kg of doxorubicin;

[0113] iii. Mice receiving a single i.p. injection of 20 mg/kg of AG1782and two hours later an i.p. injection of 20 mg/kg of doxorubicin;

[0114] iv. Mice receiving a single i.p. injection of 20 mg/kg of AG126and two hours later an i.p. injection of 20 mg/kg of doxorubicin.

[0115] The % mortality in each of the above groups was determined byscoring the number of dead mice every day until 10 days after injection.

[0116] As seen in FIG. 10, in the group of mice receiving doxorubicinalone (i), 90% of the mice were dead within a week after injection. Inthe other groups of mice receiving the tyrphostins before theadministration of doxorubicin, the % mortality was reduced to between50% (iii) to only 10% (ii).

II. Reduction of the Toxic Effect of Various Harmful Agents on Organs(Kidneys, Liver, Intestines, Heart) of Treated Mice by TyrphostinsExample 10

[0117] Groups of CD1 mice, each comprising three mice were injected i.p.with a dose of 14 mg/kg of cisplatin. The mice in each of the groupsexcept for one were injected with a single i.p. injection an tyrphostinat a dose of 10 mg/kg two hours prior to the injection of the cisplatin.In order to assess the toxic effect of cisplatin on the kidneys of thetreated mice (nephrotoxicity), the level of creatinine was determined inthe serum of each mouse four days after administration of the cisplatinusing a commercial kit by Sigma.

[0118] As seen in FIG. 5, the creatinine level of about 2 mg/dl detectedin the serum of mice receiving cisplatin alone, indicates that there isabout a 50% reduction in the kidney function of these mice. Againstthis, the level of creatinine measured in the serum of many of the micereceiving an injection of an tyrphostin before the cisplatin injection,was lower than the creatinine levels in the serum of mice receivingcisplatin alone. This indicates that injection of the tyrphostins priorto the cisplatin injection reduces the toxic effect of cisplatin onkidney function. The reducing effect on the nephrotoxicity of cisplatinvaried between the different tyrphostins. For example, AG1824, AG1744,AG1751, AG1714, AG1823 AG1782, AG1801, markedly reduced thenephrotoxicity of cisplatin bringing the creatinine levels in the serumto those of control mice.

Example 11

[0119] CD1 female mice were injected either with cisplatin alone or witha single injection comprising either the tyrphostin AG1843 or AG1714 twohours before injection of cisplatin and the level of the creatinine intheir serum was tested as explained above.

[0120] As seen in Table 1 below and in agreement with the results ofExample 10 above, cisplatin alone had a nephrotoxic effect on the miceas seen by their high serum creatinine levels (1.53). This toxic effectof cisplatin was significantly reduced (to between 0.45-0.70) byinjecting the tyrphostins two hours before cisplatin injection. In thisexample, the effect of tyrphostin AG1843 administered at a low dose (5mg/kg,) was more effective than the administration of a high dose (20mg/kg) of the same tyrphostin. TABLE 1 Groups Creatinine mg/dl (mean ±SD) Control (Cosolvent) 0.46 ± 0.01 Cisplatin 14 mg/kg i.p. 1.53 ± 0.04AG1843 5 mg/kg i.p. (−2 h) + 0.45 ± 0.06 Cisplatin AG1843 20 mg/kg (−2h) + 0.70 ± 0.14 Cisplatin AG1714 20 mg/kg (−2 h) + 0.58 ± 0.06Cisplatin

Example 12

[0121] CD1 mice were injected (i.p.) with cisplatin (14 mg/kg) or firstwith AG1714 (10 mg/kg) and two hours after with cisplatin as explainedabove. Mice in the control group received injections of the vehicle only(a stock solution of ethanol: chemaphore (50:50) diluted with saline).Four days after administration of the cisplatin, several parametersindicating nephrotoxicity and liver injury were measured in the serumsof each of the mice 4 days after cisplatin administration. Theparameters showing nephrotoxicity of cisplatin were creatinine and bloodurea nitrogen (BUN) and the parameters indicating damage to the liverwere the hepatic transaminases alanine-transaminase (ALT) and aspartictransaminase (AST) measured by standard methods. Each group consisted ofthree mice and the results are shown as mean levels of the measuredparameter±SD.

[0122] As seen in FIG. 6, administration of cisplatin alone to the miceresulted in elevated levels of creatinine (FIG. 6A) and BUN (FIG. 6B).Table 2 below shows that the levels of ALT and AST were also elevated inthese mice. These results indicate both nephrotoxicity as well as damageto the liver of the mice as a result of the administration of cisplatin.As seen in FIG. 6 and Table 2, administration of AG1714 two hours beforeinjection of the cisplatin, resulted in a reduced level of all fourmeasured parameters to their normal level as measured in the controlmice. TABLE 2 Prevention of cisplatin - induced kidney and liver injuryby AG1714 ALT AST Treatment u/L u/L Control (vehicle) 30 ± 4.7 58 ± 1.5Cisplatin (14 mg/kg) 73.5 ± 2.1   209 ± 16.0 Cisplatin (14 mg/kg) + 36 ±3.6  70 ± 14.0 AG1714 (20 mg/kg)

Example 13

[0123] CD1 mice were injected with 14 mg/kg of cisplatin or first with asingle i.p. injection of the tyrphostin AG1714 and two hours later aninjection of 14 mg/kg of cisplatin. Histopathological analysis of thekidneys and small intestines of the non-treated and treated mice wasperformed using slices from formaline fixed tissues stained withhematoxylin eosin.

[0124] As seen in FIG. 7, kidneys of mice receiving cisplatin aloneshowed large proteinaceous plaques in the proximal tubals (7B) as wellas the appearance of granular material in the columnar epithel. Againstthis, kidneys of the mice receiving AG1714 injection prior to thecisplatin administration (7C) showed no damage and their structure wassimilar to that of control mice (7A).

[0125] As also seen in FIG. 7, the small intestine of mice receivingcisplatin alone (7E) showed severe necrosis and disintegration of thecolumnar intestinal epithelial cells as compared to the small intestinesof mice receiving AG1714 prior to the cisplatin administration (7F)which showed a normal structure similar to that seen in the smallintestine of non-treated mice (7D).

Example 14

[0126] In order to test the protective effect of AG1714 againstdoxorubicin-induced cardiotoxicity, 7 day primary rat cardiomyocytescultures were prepared according to a standard procedure (described in:In vitro Toxicology, Model System and Methods, Eds. C. McQueen, TelfordPress New Jersey, 1990, pg. 163, Primary cultures of neonatal ratmyocardial cells, and Kessler-Icekson, G., J. Mol. Cell Cardiol,20:649-755, 1988).

[0127] The cardiomyocyte cultures were divided into the following fourgroups receiving:

[0128] i. Vehicle only (control);

[0129] ii. AG1714 as a final concentration of 20 μM;

[0130] iii. Cells receiving doxorubicin at a concentration of 10 μM; and

[0131] iv. Cells receiving AG1714 at a final concentration of 20 μMfollowed one hour later by the addition of doxorubicin (10 μM).

[0132] The rate of beats of clusters of cardiomyocytes (“mini-hearts”)was determined 24 hours after beginning of incubation.

[0133] As seen in Table 3, in the cell cultures incubated withdoxorubicin alone, the number of beats per minute was significantlyreduced to about 20% of that of the control cultures incubated with thevehicle only. Against this, in cultures incubated with doxorubicintogether with AG1714, the number of beats per minute was similar to thenumber of beats per minute in the control cultures. Similar results (notshown) were seen in the above cardiomyocyte cultures 16 hours and 40hours after incubation. Thus, incubation of the cultures with AG1714 onehour before the addition of doxorubicin prevented the cardiotoxic effectof doxorubicin on the cell cultures. TABLE 3 Protective effect of AG1714against Doxorubicin - induced cardiotoxicity, in vitro Cellular beatingTreatment (beats/min) Control (vehicle) 78 ± 2.0 AG1714 (20 uM) 68 ± 2.2Doxorubicin (10 uM) 15 ± 1.3 Doxorubicin + 62 ± 2.6 AG1714

Example 15

[0134] The FAS antigen is a cell surface protein belonging to the twofactor/nerve (growth factor receptor family and have been shown tomediate apoptosis (Itoh, N. et al., Cell, 66:233-243(1991)).Intraperitoneal administration of an anti-FAS antibody into mice wasshown to cause severe damage of the liver by apoptosis.

[0135] In order to test the effect of the tyrphostin AG1801 and AG1714on the anti-FAS antibody induced hepatotoxicity, Balb/C mice wereinjected i.p. either with AG1801 (5 mg/kg) or with AG1714 (5 mg/kg) andtwo hours later with an i.p. injection of an anti-FAS antibody at a doseof 5 μg/mouse. Another group of mice received only an injection ofanti-FAS antibody with no prior treatment with the tyrphostins. Fivehours after injection of the anti-FAS antibody, the animals weresacrificed and the level of two transaminases AST and ALT in the serumof the sacrificed mice was determined: a high level of thesetransaminases indicating hepatotoxicity (Ogasaware, J., et al., Nature,364:806-809, 1993).

[0136] As seen in FIGS. 8 and 9, the level of AST and ALT in the serumof mice treated with the anti-FAS antibody alone was very highindicating FAS antibody induced hepatotoxicity.

[0137] Against this, the level of the two transaminases AST and ALT inserum of mice which were treated with AG1801 (FIG. 8) or with AG1714(FIG. 9) prior to the anti-FAS injection was significantly lower. Thus,administration of the tyrphostins to mice results in their protectionagainst hepatotoxicity induced by the anti-FAS antibody.

Example 16

[0138] T-cell mediated hepatotoxicity is a complication of variousdisorders and diseases such as Hepatitis B virus infection. Suchhepatotoxicity can be induced by injection of Conavalin A (Con A). Theeffect of AG1714 on Con A induced hepatotoxicity on mice was determinedin the following way (see Tiegs, G. et al., J. Clin. Invest.,90:196-203, 1992).

[0139] CD1 mice were divided into the following groups:

[0140] i. Mice receiving injection of the vehicle only (control);

[0141] ii. Mice receiving i.p. injection of AG1714 (10 mg/kg);

[0142] iii. Mice receiving an i.v. injection of Con A (35 mg/mouse); and

[0143] iv. Mice receiving, an i.p. injection of AG1714 (10 mg/kg) andtwo hours later an i.v. injection of Con A (35 mg/mouse).

[0144] 6 hours after injection of Con A to the mice, serum levels of theliver enzymes AST and ALT of the treated mice were determined asexplained above. Increase of serum levels of these enzymes reflect liverinjury.

[0145] As seen in FIG. 10, while administration of AG1714 alone had noeffect on the mice, injection of Con A caused significant hepatotoxicityin the mice. Administration of AG1714 prior to Con A administrationsignificantly reduced the Con A induced hepatotoxicity in these mice.

Example 17

[0146] It is today known that liver damage may be induced by variousimmune mechanisms such as cytokines as well as by a variety of otheragents known to exert an apoptotic effect (e.g. alcohol or paracetemol).A model for such liver injury induced by apoptosis was developed inwhich the hepatic damage is induced in vivo by the injection ofgalactosamine and TNF-α (see Leist, M., et al., J. Immunol.,153:1778-1783, 1994).

[0147] The effect of AG1714 on TNF-induced liver damage in mice wasdetermined as follows.

[0148] Mice were divided into the following four groups:

[0149] i. Mice receiving an injection of the vehicle alone;

[0150] ii. Mice receiving 1 i.p. injection of AG1714 (10 mg/kg);

[0151] iii. Mice receiving an i.v. injection of human TNF-α (0.25μg/mouse) in combination with galactosamine (GaIN) (18 ml/mouse, i.p.);and

[0152] iv. Mice receiving the treatment of group iii above together withan injection of AG1714 (10 mg/kg) injected i.p. two hours prior toTNF/GaIN injection. 7 hours after injection of the TNF/GaIN, the serumlevels of the liver enzymes AST and ALT in the serum of the varioustreated mice was determined as described above.

[0153] As can be seen in FIG. 11, injection of AG1714 prior to theadministration of TNF/GaIN to the mice significantly reduced thehepatoxicity induced by the TNF/GaIN administration. The tyrphostins maytherefore be useful in the reduction of liver damage induced by variousimmune mechanisms.

III. Reduction of the Toxic Effect of Various Harmful Agents onNucleated Bone Marrow Cells (Myelotoxicity) and Lymphocytes(Lymphotoxicity) by Tyrphostins Example 18

[0154] CD1 mice were divided into the following groups:

[0155] i. Mice receiving an i.p. injection of doxorubicin (10 mg/kg)alone;

[0156] ii. Mice receiving an i.p. injection of AG1714 (20 mg/kg) and twohours later an i.p. injection of doxorubicin (10 mg/kg); and

[0157] iii. Mice receiving one injection of the vehicle and two hourslater an i.p. injection of doxorubicin (10 mg/kg).

[0158] 3 days later, the number of bone marrow nucleated cells andcolony forming units (CFU) in the femurs of the treated mice wasdetermined (Nikerich, D. A., et al., J. Immunopharmacol., 8:299-313,1986). Each group contained 3 mice and the results arc expressed as ameans±SD.

[0159] As seen in FIG. 12, injection of doxorubicin to mice resulted 3days later in myelotoxicity as manifested by 47% reduction in the numberof nucleated cells (FIG. 12A) and 55% reduction in CFU (FIG. 12B). Micewho were pretreated with AG1714 two hours prior to doxorubicin injectionwere fully protected against its myelotoxicity effects.

Example 19

[0160] The effect of AG1714 on myelotoxicity induced by doxorubicin atdifferent doses was investigated. Mice were treated as described inExample 18 above. The number of femur nucleated bone marrow cells in thetreated mice was determined three days (FIG. 13A) and at various times(FIG. 13B) post doxorubicin administration.

[0161] As seen in FIG. 13A, administration of doxorubicin to mice at aconcentration of 10 mg/kg and 15 mg/kg induced myelotoxicity in thetreated mice. Pretreatment with AG1714 reduced the doxorubicin inducedmyelotoxicity significantly.

[0162] As seen in FIG. 13B, pretreatment of mice administered withdoxorubicin with AG1714, resulted also in a rapid recovery of bonemarrow cells after doxorubicin administration. 5 days after treatmentwith doxorubicin (15 mg/kg) alone, the number of bone marrow cells inthe femurs of the treated mice was still significantly reduced.Pretreatment of the mice with AG1714 prior to the doxorubicinadministration, fully reconstituted their bone marrow at that time.

Example 2 CD1 mice received either a single i.p. injection ofdoxorubicin (10 mg/kg) or first single i.p. injection of AG1714 (20mg/kg) and two hours later, an i.p. injection of doxorubicin (10 mg/kg).72 hours after doxorubicin administration the mice were sacrificed andthe weight of the spleen and thymus of the mice was determined asparameters of lymphotoxicity. Each experimental group consisted of threemice and the results arc mean no. of cells±SD. As control, the micereceived an injection of the vehicle only instead of doxorubicin.

[0163] As seen in FIG. 14 below, the administration of doxorubicin alonecaused a significant reduction in the weight of spleens (FIG. 14A) andthymuses (FIG. 14B) of the mice. This myelotoxic and lymphotoxic effectof doxorubicin was significantly reduced by the administration of AG1714to the mice two hours before administration of the doxorubicin.

Example 21

[0164] CD1 mice were injected with a single i.p. injection ofcyclophosphamide at a dose of 50 mg/kg and divided into the followinggroups;

[0165] i. Mice receiving a single i.p. injection of the vehicle only twohours before administration of the cyclophosphamide;

[0166] ii. Mice receiving a single i.p. injection of AG1714 (20 mg/kg)two hours before cyclophosphamide;

[0167] iii. Mice receiving a single i.p. injection of AG1801 (2.5 mg/k,)two hours before cyclophosphamide administration.

[0168] 72 hours after cyclophosphamide administration the mice weresacrificed and the nucleated cells in the bone marrow, spleen and thymusof the mice were counted. Each experimental group consisted of threemice and the results shown are the mean number of cells in a group±SD.

[0169] As seen in Table 4A and B, administration of cyclophosphamidecaused a significant reduction in the number of nucleated cells in thebone marrow, spleen and thymus indicating myelotoxic and lymphotoxiceffect of cyclophosphamide. Administration of the tyrphostins AG1714 orAG1801 prior to the administration of cyclophosphamide, resulted in areduction in the myelotoxic and lymphotoxic effect of cyclophosphamidein the above three organs. The number of nucleated cells in these organswas higher than in organs of mice receiving cyclophosphamide alonealthough the level of the nucleated cells in the bone marrow, spleen andthymus of the mice treated with the tyrphostins did not reach controllevel TABLE 4a and b AG1714 and AG1801 prevent Cyclophosphamide -induced myelotoxicity and lymphotysis Nucleated cells in Spleen ThymusTreatment bone marrow (× 10⁶) (mg) (mg) AG1714 (20 mg/kg) Vehicle − +− + − + 17.3 ± 1.33 17.9 ± 1.1 109 ± 20.0 112 ± 11.3 75 ± 4.0 71 ± 9.8Cyclophosphamide  6.2 ± 0.31 11.2 ± 0.6 52 ± 2.5 87 ± 2.0 39 ± 6.1 57 ±3.6 (50 mg/kg) AG 1801 (2.5 mg/kg) Vehicle − + − + − + 17.3 ± 18.6 ± 1.0109 ± 20.0  97 ± 1.52 75 ± 4.0 74 ± 7.9 1.33 Cyclophosphamide  6.2 ±0.31 10.9 ± 0.9 52 ± 2.5  69 ± 0.58 39 ± 6.1 57 ± 5.6 (50 mg/kg)

Example 22

[0170] CD1 female mice were divided into the following groups:

[0171] i. A control group receiving a single i.p. injection of thevehicle only (co-solvent: propylene carbonate-cremophor-saline);

[0172] ii. Mice receiving a single i.p. injection of 5-fluorouracil (5FU) at a dose of 80 mg/kg;

[0173] iii. Mice receiving a single i.p. injection of AG1714 at a doseof 20 mg/kg;

[0174] iv. Mice receiving a single i.p. injection of AG1801 at a dose of2.5 mg/kg;

[0175] v. Mice receiving a single i.p. injection of AG1843 at a dose of5 mg/kg;

[0176] vi. Mice receiving a single i.p. injection of AG1714 (20 mg/kgand two hours later a single i.p. injection of 5 FU (80 mg/kg);

[0177] vii. Mice receiving a single i.p. injection of AG1801 (2.5 mg/kg)and two hours later a single i.p. injection of 5 FU (80 mg/kg); and

[0178] viii. Mice receiving a single i.p. injection of AG1843 (5 mg/kg)and two hours later a single i.p. injection of 5 FU (80 mg/kg).

[0179] Five days after the administration of 5 FU to the mice, the micewere sacrificed and the number of nucleated cells in the bone marrow ofone femur, were counted. Each group of mice consisted of two or threemice and the results shown are the mean number of cells counted±SD.

[0180] As seen in FIG. 15, administration of 5 FU to the mice resultedin a decrease in the number of nucleated cells in the bone marrow ofthese mice. The administration of the AG1714 before 5 FU administrationto the mice, resulted in a significantly lower reduction in the numberof cells in the bone marrow. Thus the AG1714 reduced the myelotoxiceffect of 5 FU in these mice.

Example 23

[0181] CD1 female mice were divided into the following groups:

[0182] i. A control group receiving a single i.p. injection of thevehicle only (co-solvent: propylene carbonate-cremophor-saline);

[0183] ii. Mice receiving a single i.p. injection of mitomycin-C at adose of 2 mg/kg;

[0184] iii. Mice receiving a single i.p. injection of AG1714 at a doseof 20 mg/kg;

[0185] iv. Mice receiving a single i.p. injection of AG1714 (20 mg/kg)and two hours later a single i.p. injection of mitomycin-C (2 mg/kg);

[0186] As described in Example 22 above, five days after theadministration of mitomycin-C to the mice, the mice were sacrificed andthe number of nucleated cells in the bone marrow of one femur, werecounted. Each group of mice consisted of two or three mice and theresults shown are the mean number of cells counted±SD.

[0187] As seen in FIG. 16, mice receiving mitomycin-C alone showed avery low count of nucleated cells in their bone marrow. Whileadministration of AG1714 alone had no effect on the mice, itsadministration prior to the administration of mitomycin-C to the miceresulted in a reduction in the myelotoxic effect of the mitomycin-C inthese mice and provided almost full protection from the harmful effectsof the mitomycin-C bringing the nucleated cell count in the bone marrowof these mice to the level of control mice receiving the cosolvent only.

Example 24

[0188] The effect of various doses of AG1801 against doxorubicin inducedmyelotoxicity in mice was determined. CD1 mice were divided into fivegroups:

[0189] i. Mice receiving the vehicle alone;

[0190] ii. Mice receiving doxorubicin (10 mg/kg) alone;

[0191] iii. Mice receiving an i.p. injection of AG1801 (0.5 mg/kg) twohours prior to doxorubicin administration;

[0192] iv. Mice receiving an i.p. injection of AG1801 (1 mg/kg) twohours prior to doxorubicin administration; and

[0193] v. Mice receiving an i.p. injection of AG1801 (2 mg/kg) two hoursprior to doxorubicin administration.

[0194] Three days after doxorubicin administrations the mice weresacrificed and the number of nucleated cells in bone marrow wasdetermined as described above.

[0195] As seen in FIG. 17, administration of AG1801 to the mice had aprotective effect against doxorubicin induced myelotoxicity which wasdose dependent.

IV Reduction of Radiation-Induced Toxicity by Tyrphostins Example 25

[0196] In order to test the protective activity of the tyrphostin AG1714in radiated mice, CD1 mice irradiated with a radiation dose of 300Rusing a cobalt source were divided into two groups:

[0197] i. Mice receiving no further treatment; and

[0198] ii. Mice receiving a single i.p. injection of the tyrphostinAG1714 at a dose of 20 mg/kg, two hours before irradiation.

[0199] A third group of mice received only a single injection of AG1714(20 mg/kg) and a fourth group of mice served as control mice which werenot treated.

[0200] At different periods of time after irradiation, mice weresacrificed and the number of cells in the bone marrow of one femur werecounted.

[0201] As seen in FIG. 18, irradiation of the mice with 300R caused adecrease in the number of cells in the bone marrow of these mice ascompared to untreated mice or mice treated only with AG1714.Reconstitution of cells in the bone marrow of the irradiated mice beganabout five days after their radiation. Treatment of the irradiated micewith AG1714 prevented the decrease in the number of cells in the bonemarrow of the irradiated mice and caused a very significant enhancementof the number of cells in their bone marrow. Thus, the tyrphostin AG1714has a protective effect against myelotoxicity caused by irradiation.

Example 26

[0202] The protective effect of the tyrphostin AG1714 againstmyelotoxicity caused by a high dose irradiation (450R) was tested asdescribed in Example 25 above except that the dose of irradiation was450R instead of 300R.

[0203] As seen in FIG. 19, the number of cells in the bone marrow ofmice irradiated with 450R was significantly reduced five days afterradiation indicating a severe myelotoxic effect of the irradiation onthese mice. Five days after irradiation reconstitution of the number ofcells in the bone marrow began but 18 days after radiation, the numberof cells in the bone marrow of the irradiated mice still remainedsignificantly lower than the number of cells in the bone marrow ofnon-treated mice or mice receiving AG1714 alone. In the bone marrow ofmice receiving AG1714 before their irradiation, myelotoxic effect wasalso seen until five days after radiation. However, after five days, asignificant reconstitution was seen in the number of cells in the bonemarrow of these mice and 18 days after irradiation, the number of cellsin the bone marrow of the irradiated mice receiving AG1714 was higherthan the number of cells in the control mice. Administration of AG1714thus enhance the reconstitution of the number of cells in the bonemarrow of mice irradiated by high dose irradiation.

Example 27

[0204] CD1 mice were irradiated with a lethal 800R dose using a cobaltsource, and divided into two groups:

[0205] i. Mice receiving irradiation only; and

[0206] ii. Mice receiving a single i.p. injection of AG1714 at a dose of20 mg/kg one hour before irradiation.

[0207] Each croup of mice consisted of 10 mice and the % mortality ineach group was tested by scoring the number of dead mice each day afterirradiation.

[0208] As seen in FIG. 20, in the group of mice receiving an 800Rirradiation, mortality of the mice began 10 days after irradiation andthe % mortality in this group reached the rate of 80% 23 days aftertheir irradiation. Against this, the % mortality in the group of micereceiving AG1714 before their irradiation was significantly reduced to20% mortality 20 days after irradiation.

[0209] The mortality in both groups did not change further until 50 daysafter irradiation.

V. The Effect of Tyrphostins on Anti-Tumor Activity of ChemotherapeuticAgents Example 28

[0210] The effect of AG1714 on the anti-tumor effect of chemotherapy wasdetermined using a variety of experimental tumor models in mice.

[0211] 2×10⁵/mouse MCA-105 fibrosarcoma cells or Lewis Lung carcinomacells and 5×10⁴/mouse B-16 melanoma cells were injected into the tailvein of C57BL mice.

[0212] Cisplatin (4 mg/kg) was injected i.p. 4 days after tumorinoculation. AG1714 (20 mg/kg) or vehicle was injected i.p. 2 hoursprior to cisplatin. Twenty-four days after tumor inoculation, mice weresacrificed, lungs weighed and the number of metastases were scored. Eachgroup contained 5 mice and the results are expressed as the means lungs'weights±SE.

[0213] As seen in FIG. 21A, cisplatin markedly inhibited the growth ofMCA-105 fibrosarcoma. AG1714 by itself had a small anti-tumor effect.Combined treatment with AG1714 and cisplatin was as effective ascisplatin alone, in suppression of the growth of the established lungmetastases of MCA-105. Cisplatin and AG1714 by themselves had a partialanti-tumor effect against Lewis lung carcinoma. Combined treatment withAG1714 and cisplatin was more effective than cisplatin also insuppression of the growth of established lung metastases of this tumor(FIG. 21B).

[0214] Cisplatin (4 mg/kg) or AG1714 (20) mg/kg) alone were noteffective against established lung metastases of B-16 melanoma (FIG.21C). Combined treatment with these agents was more effective thantreatment with each agent alone.

Example 29

[0215] The effect of doxorubicin and AG1714 on SK-28 human melanomatumor xenografts in nude mice was determined as follows:

[0216] CD1 athymic mice (nu/nu) were inoculated i.v. with 4×10⁵/mouseSK-28 human melanoma. Doxorubicin (4 mg/kg) was injected i.p. 4 daysafter tumor inoculation. AG1714 (20 mg/kg) or vehicle were injected 2hours prior to doxorubicin. The weights of the treated mice lungs andnumber of metastases in the lungs were determined 24 days after tumorinoculation. Each group contained 5 mice and the results are expressedas means±SE.

[0217] As seen in FIG. 22, doxorubicin markedly suppressed the growth ofthe SK-28 tumor whereas AG1714 by itself had a small anti-tumor effect.Pretreatment with AG1714 did not impair the effect of doxorubicin alonein the suppression of the growth of this tumor.

Example 30

[0218] Athymic mice were inoculated subcutaneously (s.c.) with humanovary carcinoma (OVCAR-3) cells (3×10⁶ cells/site). Cisplatin (4 mg/kg)was injected 4 days after tumor inoculation. AG1714 (20 mg/kg) orvehicle was injected i.p. 2 hours prior to cisplatin. Twenty days posttumor inoculation the short (S) and long (L) diameters of the tumor weremeasured and the tumor volume (V) was calculated according to theformula:  $V = {\frac{\lbrack {S^{2} \times L} \rbrack}{2}.}$

[0219]  Each group contained 5 mice and the results are expressed as themeans±SE.

[0220] As seen in FIG. 23, cisplatin had a marked anti-tumor effect inthe mouse bearing OVCAR-3 tumors whereas AG1714 itself was lesseffective. Administration of AG:1714 did not impair the chemotherapeuticeffect of cisplatin.

[0221] As seen in FIG. 24, the difference in the diameter of the tumorvolume of tumors in mice receiving repeated injections of cisplatin andAG1714 as compared to the volume of tumors in mice receiving repeatedinjections of cisplatin alone or repeated injections of AG1714 alone.

Example 31

[0222] The effect of AG1714 on high dose doxorubicin efficacy wasmeasured by determining the mortality of B-16 melanoma bearing mice andthe number of metastases in their lung's or the lung weight.

[0223] Mice were injected i.v. with 2×10⁵ B-16 melanoma cells and 4 daysafter tumor inoculation each mouse received a single i.p. injection ofdoxorubicin (4 mg/kg, or 8 mg/kg). Two hours prior to the doxorubicininjection, each mouse was injected i.p. with AG1714 at a dose of 20mg/kg.

[0224] As seen in Table 5A and B showing two experiments carried out asexplained above, administration of low dose doxorubicin resulted in lowmortality of the mice and in a significant therapeutic effect. High doseadministration of doxorubicin resulted in a much more significanttherapeutic effect but resulted in a high mortality rate of the treatedmice. AG1714 administered alone showed some therapeutic effect initself. The combined administration of doxorubicin at a high dosetogether with AG1714 resulted in the high therapeutic effect of the highdoxorubicin, however, the mortality of the mice were significantlyreduced. TABLE 5 Effect of AG1714 on high dose doxorubicin efficacy andmortality in B-16 melanoma bearing mice A. No. of lungs' metastasesMortality Treatment (+SD) (dead/total) Vehicle 29 ± 9  0/4 AG1714 (20mg/kg) 16 ± 5  0/4 Doxoruhicin (4 mg/kg) 7 ± 4 0/4 Doxorubicin (4mg/kg) + 7 ± 6 0/4 AG1714 (20 mg/kg) Doxorubicin (8 mg/kg) 1     3/4(75%) Doxorubicin (8 mg/kg) + 2 ± 1 0/4 AG1714 (20 mg/kg) No tumor — 0/4B. Lungs' weight Mortality Treatment (mg + SEM) (dead/total) Vehicle 976 ± 142 0/6 AG1714 (20 mg/kg)  870 ± 178 0/6 Doxorubicin (4 mg/kg)306 ± 12 0/6 Doxorubicin (4 mg/kg) + 303 ± 28 0/6 AG1714 (20 mg/kg)Doxorubicin (8 mg/kg) —     7/8 (88%) Doxorubicin (8 mg/kg) + 246 ± 10    2/8 (25%) AG1714 (20 mg/kg) No tumor 232 ± 12 0/3

[0225] Thus the combined administration of AG1714 together withdoxorubicin resulted in the intensification of the chemotherapeuticeffect of doxorubicin. The significant therapeutic effect of the highdose doxorubicin administered with AG1714 could be achieved while thehigh mortality rate of the high dose doxorubicin was neutralized by thecombined administration.

[0226] Such an intensification effect could also be seen in mice bearingMCA-105 fibrosarcoma tumors and treated with either doxorubicin alone(Table 6A) or cisplatin alone (Table 6B) or with a combination of eachof the above with AG1714. TABLE 6 Effect of AG1714 on high dosedoxorubicin efficacy and mortality in MCA-105 fibrosarcoma bearing miceLungs' weight Mortality Treatment (mg + SEM) (dead/total) A. Vehicle 908± 107 0/8 AG1714 (20 mg/kg) 632 ± 74  0/6 Doxorubicin (4 mg/kg)  47 ±119 0/5 Doxorubicin (4 mg/kg) + 494 ± 137 0/5 AG1714 (20 mg/kg)Doxorubicin (8 mg/kg) 170 ± 4  5/8   (62.5%) Doxorubicin (8 mg/kg) + 172± 6  2/8 AG1714 (20 mg/kg) (25%) No tumor 161 ± 12  0/4 B. Vehicle 982 ±102 0/6 AG1714 (20 mg/kg) 656 ± 127 0/6 Doxorubicin (4 mg/kg) 420 ± 69 0/6 Doxorubicin (4 mg/kg) + 216 ± 33  0/6 AG1714 (20 mg/kg) Doxorubicin(8 mg/kg) 186 ± 16  5/7 (71%) Doxorubicin (8 mg/kg) + 167 ± 5  2/6AG1714 (20 mg/kg) (33%) No tumor 161 ± 6  0/6

Example 32

[0227] The effect of AG1801 on the anti-metastatic activity of cisplatinon established lung metastases of MICA-105 fibrosarcoma bearing, micewas determined as follows:

[0228] C57BL mice were divided into the following groups:

[0229] i. Mice injected i.v. at the age of 6 weeks with 2×10⁵fibrosarcoma MCA105 cells and 4 days' later, receiving, an injectioncomprising the vehicle only;

[0230] ii. Mice injected i.v. at the age of 6 weeks with 2×10⁵fibrosarcoma MCA105 cells and 4 days' later, injected with 10 mg/kg, ofcisplatin,

[0231] iii. Mice injected i.v. at the age, of 6 weeks with 2×10⁵fibrosarcorna MCA105 cells and 4 days' later, injected with 10 mg/kg of,doxorubicin;

[0232] iv. Mice injected i.v. at the age of 6 weeks with 2×10⁵fibrosarcoma MCA105 cells and 4 days' later, injected with 5 mg/kg ofAG1801;

[0233] v. Mice injected i.v. at the age of 6 weeks with 2×10⁵fibrosarcoma MCA-105 cells and 4 days' later, injected with 5 mg/kg ofAG1801 and 2 hours' later, with 10 mg/kg of cisplatin;

[0234] vi. Mice injected i.v. at the age of 6 weeks with 2×10⁵fibrosarcoma MCA-105 cells and 4 days' later, injected with 5 mg/kg ofAG1801 and 2 hours' later, with 10 mg/kg of doxorubicin; and

[0235] vii. Non treated C57BL mice.

[0236] As seen in FIG. 25A and 25B, the weight of the lungs of the tumorbearing mice were significantly higher than that of the control mice.While injection of AG1801 alone had no affect, the injection ofcisplatin (FIG. 25A) or doxorubicin (FIG. 25B) reduced the weight of themice's lungs to a weight similar to that of control non tumor bearingmice. The combined injection of cisplatin and AG1801 (FIG. 25A) and thecombined injection of doxorubicin and AG1801 (FIG. 25B) resulted inreduction of tumor load in the lungs of the mice similar to thereduction in the tumor load of mice receiving the cisplatin alone.

[0237] The results of the above four examples clearly show thatadministration of tyrphostins to tumor bearing, mice together withcisplatin does not affect the antitumor affect of the cisplatin and insome cases eve;<increases it.

Example 33

[0238] The effect of cisplatin and AG1801 on SK-28 human melanoma tumorxenografts in nude mice was determined as explained in Example 29 above.

[0239] As seen in FIG. 26, the administration of cisplatin alone reducedthe weight of the lungs of the tumor bearing mice to an extent whileadministration of AG1801 had no therapeutic effect in itself. Combinedadministration of AG1801 with cisplatin significantly reduced the rateof lungs of the treated mice and thus the administration of AG1801 priorto cisplatin administration enhanced its therapeutic effect.

[0240] The above examples clearly show that administration oftyrphostins to tumor bearing mice together with cisplatin or doxorubicindoes not impair the anti-tumor effect of the therapeutic agents and insome cases even increases it. Therefore the tyrphostins could be usefulin potentiating and intensifying the chemotherapeutic effect of suchagents.

VI. The Effect of AG1714 on the Viability of Various Cells in vitro wasDetermined as Follows Example 34

[0241] Murine bone marrow nucleated cells were prepared by gradientcentrifugation on histopaque. The cells were then divided into twogroups:

[0242] i. Cells incubated for one hour in growth medium alone (control);

[0243] ii: Cells incubated for one hour in growth medium with theaddition of various concentrations (5 μM; 10 μM or 20 μM) of thetyrphostin AG1714.

[0244] Cells were then plated on semi-solid agar and the number ofcolony forming units (CFU) was determined after a 14 day incubationperiod (see: Nikerich et al., supra, 1986).

[0245] As seen in FIG. 27, the plating, efficiency (determined by thenumber of CFU per 10⁵ nucleated cells) of the cultures to which AG1714was added was significantly enhanced as compared to that of the cellsgrown in growth medium alone. Thus, the tyrphostin AG1714 enables longerpreservation of bone marrow cell culture.

Example 35

[0246] Thymocytes were prepared from thymuses of young CD1 mice at aconcentration of 3×10⁶ cell/ml in RPNI1640 growth medium containing 5%FCS. The cells were divided into two groups:

[0247] i. A Cells grown in the presence of growth medium alone; and

[0248] ii. Cells grown in growth medium with the addition of AG1714.

[0249] The cell viability of the thymocytes in each of the abovecultures was determined using the trypsin blue exclusion test at variousperiods of time after the addition of AG1714 to the cells.

[0250] As seen in FIG. 28, addition of AG1714 to the thymocytes inculture significantly enhanced their viability.

Example 36

[0251] Primary rat myocyte cultures were prepared from rats as describedin Kessler-Iccksan, G., J. Mol. Cell. Cardiol., 20:649-755, 1988.Cardiomyocytes were grown in culture for seven days and then thecultures were divided into two groups:

[0252] i. Cultures grown in growth culture alone;

[0253] ii. Cultures grown in growth culture with the addition of 20 μMof the tyrphostin AG 1714.

[0254] The number of beats per minute of the myocyte clusters in eachculture was determined 16 hours and 40 hours after the addition of thetyrphostins to the cells.

[0255] As seen in FIG. 29, the number of beats per minute in thecardiomyocyte cultures to which the tyrphostin AG1714 was added weresignificantly higher than the number of beats per minute in the culturesgrown in the growth medium alone. AG1714 enhanced the viability of thecardiomyocytes in culture.

VII. Toxicity Study of Tyrphostins Example 37

[0256] In order to study the toxic effects of the tyrphostins AG1714 andAG1801, the tyrphostins were injected to ICR mice 3 times a week over aperiod of five weeks. Each injection contains 20 mls. of the tyrphostinswhich was prepared in the vehicle propylene carbonate:cremophore (40/60)diluted 1:20 with saline/bicarbonate.

[0257] In order to test the toxic effect of the cumulativeadministration of the tyrphostins, various parameters of the injectedmice were determined including their weight, weight of their internalorgans (thymus, spleen, kidney), full blood chemistry, number of bonemarrow cells, etc.

[0258] As seen in Table 7 below, injection of the tyrphostins AG1714 andAG1801 caused no significant changes in the parameters tested in theinjected mice as compared to the same parameters in control miceinjected with saline or the vehicle only. Therefore, no toxic effects ofthe tyrphostins were apparent following their cumulative administrationto mice. In addition, injection of the tyrphostins caused no mortalityof the mice. TABLE 7 Subchronic intraperitoneal administration of AG1714and AG1801 (ICR female mice, injection 3 times a week, 5 weeks) Control1 Control 2 AG1714 Ag1801 Saline Vehicle 20 mg/kg × 15 5 mg/kg × 15Weight (g) 29.3 ± 3.1  28.6 ± 2.3  29.1 ± 1.7  Thymus (mg) 50.2 ± 7.7 53.8 ± 12.3  57 ± 9.3 Spleen (mg) 118 ± 27  136 ± 16   119 ± 19.6 Kidney(mg) 176 ± 21  162 ± 12  164 ± 9.8  Leukoc. (k/ul) 6.1 ± 2.3  5.5 ± 0.984.9 ± 2.3 6.8 ± 3.3 Lymph. (%) 88.1 ± 2.35 83.3 ± 4.12 81.5 ± 2.35 80.4± 6.1  Neutr. (%) 4.2 ± 1.4 4.2 ± 1.2 5.3 ± 1.2 Monoc. (%) 10.6 ± 2.6 11.9 ± 1.3  12.3 ± 5.2  Eosin.. (%) 0.2 ± 0.1  0.1 ± 0.05  0.3 ± 0.23Basoph. (%) 0.22 ± 0.08 0.24 ± 0.11  0.3 ± 0.07 RBC (M/ul)  8.4 ± 0.33 9.0 ± 0.33 8.4 ± 0.3 HGB (g/dl) 14.0 ± 2.5  14.2 ± 0.5  14.9 ± 0.5614.5 ± 0.67 PLT (K/ul) 1236 ± 176  1229 ± 12.7  1475 ± 252  1309 ± 304 Glucose (mg/dl)  147 ± 11.3 177 ± 24  144 ± 17   177 ± 20.5 BUN (mg/dl) 22 ± 4.4  21 ± 2.9 25.2 ± 3.3  24.2 ± 2.2  Creatinine (mg/dl) 0.4 ± 0.00.42 ± 0.04 0.46 ± 0.05 0.42 ± 0.04 Total Prot. (g/dl)  5.4 ± 0.17 5.6 ±0.2 5.8 ± 0.2  5.6 ± 0.08 Albumin (g/dl) 3.1 ± 0.0  3.1 ± 0.09  3.2 ±0.13  3.1 ± 0.07 Calcium (mg/dl) 9.9 ± 0.4  9.9 ± 0.26 9.9 ± 0.3  9.8 ±0.36 In. Phosph. (mg/dl) 8.5 ± 0.7  8.0 ± 0.75  8.4 ± 0.43 7.9 ± 1.1Uric acid (mg/dl)  1.1 ± 0.21  2.0 ± 0.44  1.8 ± 0.18  2.0 ± 0.36 TotalBilirub. (mg/dl) 0.23 ± 0.06 0.2 ± 0.0 0.2 ± 0.0 0.2 ± 0.0 Alk. Phosph.(U/l)  110 ± 23.8 79 ± 17   67 ± 12.8 69.6 ± 0.26 LHD (U/l) 545 ± 157996 ± 235 960 ± 226 972 ± 238 GOT (U/l) 67 ± 11 71.4 ± 6.5  70 ± 18 68 ±12 GPT (U/l)  30 ± 4.6 28.8 ± 3.1  25.6 ± 4.2   28 ± 5.5 CPK (U/l) 461 ±179 604 ± 115 659 ± 486 582 ± 127 Amylase (U/l) 2451 ± 176  2211 ± 352 2392 ± 230  2169 ± 222  Cholester. (mg/dl) 115 ± 25  98.8 ± 17.5  99 ±5.7  90 ± 6.1 Triglycer. (mg/dl) 216 ± 84   125 ± 17.9 155 ± 54  138 ±50  Nuclear BM cells × 10⁶ 16.3 ± 0.7  15.2 ± 2.0  18.6 ± 3.6  16.5 ±0.87 Colonies (in femur) 12790 ± 1620  17600 ± 2590  15690 ± 2040 Clusters (in femur) 8170 ± 697  15000 ± 2140  13410 ± 1740 

VIII. Inhibition of Immune Specific Activities Example 38

[0259] Murine peritoneal exudate cytotoxic lymphocytes (PEL) capable ofspecific lysis of EL-4 cells were prepared and tested as described inLavy, R. et al., J. Immunol. 154:5039-5048, 1995. The EL-4 cells weredivided into cells which were incubated with PELs alone, cells whichwere incubated with AG1714 (20 μM) and then with PELs and cells whichwere incubated with AG1714 (50 μM) and then with PELs. The lysis of thetarget EL-4 cells was determined using the specific ⁵¹Cr release assay.

[0260] As seen in FIG. 30, addition of AG1714 reduced the specificcytotoxic activity of PELs against the target EL-4 cells. The inhibitiveeffect of AG1714 was dose dependent.

[0261] The above results indicate that tyrphostins may be useful forreducing immune mediated rejection of transplants, in cases ofautoimmunity and in conditions involving specific immune activityagainst cells (e.g. anti-CD4 cells such as in the case of AIDS).

IX. Anti-Inflammatory Effect of Tyrphostins

[0262] One of the main complications of an inflammatory reaction in anindividual is the development of septic shock which results from overeffects of the immune reaction which is mediated mainly by cytokinessuch as TNF and IL-1. The involvement of tyrphostins in TNF and IL-1related immune responses was determined as follows:

Example 39

[0263] A. Incubation of mouse fibroblastic cells with TNF-α result inapoptosis of these cells. In order to test the effect of tyrphostins onthe TNF-α-induced apoptosis of the cells, A9 mouse fibroblastic cellswere incubated for two hours with cycloheximide (50 μg/ml) after whichthe various tyrphostins were added at a final concentration of 20 μMexcept AG1801 that was added at the final concentration of 5 μM.Following one hour incubation with the tyrphostins, TNF-α was added tothe cells at a concentration of 0.2 ng/ml and the cells were incubatedfor an additional ten hours. At the end of ten hours, the cell cultureswere analyzed by FACS using propidium iodide and the extent of apoptosisin each of the cell cultures (seen as DNA fragmentation) was determined(see Novogrodsky, A., et al, Science, 264:1319-1322, 1994).

[0264] As seen in Table 8 below, addition of the various tyrphostins tothe mouse fibroblastic cells incubated with TNF, reduced the apoptopiceffect of TNF significantly. The extent of the reduction of theapoptopic effect of TNF by the various tyrphostins was slightlydifferent. TABLE 8 Prevention of TNF-α-induced apoptosis of mousefibroblastic cells (A-9) by tyrphostins Apoptosis (%) TNF-α (0.2 ng/ml)Tyrphostins* − + None (vehicle) 9.3 28.7 AG1714 4.5  9.9 AG1801 6.6 10.2AG126 8.3 19.4 AG1802 7.6 17.2

[0265] B. As seen in Table 9, cell cycle analysis of the above cellcultures showed that treatment of the cells with TNF-α induced a G₁arrest. There was a direct correlation between the tyrphostins'reduction of the apoptotic effect caused by TNF and their ability toprevent G₁ arrest in the cell cultures. TABLE 9 Prevention by AG1714 ofTNF-α-induced Go/G1 arrest and apoptosis in mouse fibroblastic cells(A-9) Cell cycle G1 S G2 + M Apoptosis % % Control 60.8 36.2 3.0 9.3TNF-α (0.2 ng/ml) 85.8  7.6 6.6 28.7  AG1714 (30 μM) 58.1 38.1 3.8 4.5TNF-α + AG1714 64.5 31.2 4.3 9.9

Example 40

[0266] The effect of tyrphostins on TNF-induced cytotoxicity wasdetermined in vitro using A-9 cells as described in Example 39 above. Inaddition, the effect of tyrphostin on TNF mediated cytotoxicity in vivowas determined in mice injected with LPS (which induces TNF toxicity invivo). E.coli LPS the concentration of 1.3 mg/mouse was injected i.p. toCD1 mice. While control mice were injected in addition with the vehicleonly, the remaining mice were injected with the various tyrphostins (20mg/kg) i.p. two hours prior to LPS injection. As seen in Table 10A, inagreement with the results shown in Example 39 above, addition oftyrphostins to the cells incubated with TNF significantly reduces thecytotoxic effect of TNF. As seen in Table 10B, most of the tyrphostinsshowed a significant protective effect against the LPS-induced toxicityin vivo which correlated with their activity in vitro. TABLE 10 Effectof tyrphostins on TNF - induced cytotoxicity (in vitro) and LPS -induced lethal toxicity (in vivo) A in vitro, (live cells, % of control)TNF AG (20 μM) None 0.05 ng/ml 0.2 ng/ml 1.0 ng/ml Control 100 41 19  6AG1714 119 102  90 62 AG1719 102 83 58 41 AG26 107 86 64 31 AG126 107 7549 33 AG1641 102 70 41 21 AG1720 104 53 26 11 B in vivo alive/total %alive mice  4/10 40 5/5 100  4/5 80 4/5 80 4/5 80 3/5 60 2/5 40

Example 41

[0267] IL-1 is one of the main mediators of septic shock in vivo. Inorder to determine the inhibitive activity of AG1714 on production ofIL-1 from cells incubated with LPS, human peripheral blood monocytes(PBM) at a concentration of 2×10⁶ cells/ml were divided into thefollowing groups:

[0268] i. Cells grown in growth medium alone;

[0269] ii. Cells grown in growth medium to which AG1714 was added (20μM);

[0270] iii. Cells grown in growth medium to which LPS was added (10mg/ml); and

[0271] iv. Cells grown in growth medium with the addition of LPS (10mg/ml) and AG1714 (20 μM). The cells were incubated for 24 hours and theamount of IL-1β was determined using the Genzyme ELISA kit.

[0272] As seen in Table 11, which shows two experiments carried out asdescribed above, addition of AG1714 to cells incubated with LPSsignificantly reduced the amount of IL-1β produced by the cells. TABLE11 Inhibition by AG1714 of LPS-induced IL-1β production in humanperipheral mononuclear cells IL-1β pg/ml AG1714 (20 μM) Exp. 1 − + None2522 1174 LPS (10 μg/ml) 10187  1230 Exp. 2 − − None  583  648 LPS (10μg/ml) 9309  970

[0273] The above results indicate that tyrphostins may be useful inreducing, the non desired IL-1 mediated effect involved in septic shock.

[0274] The results of the above three examples indicate that tyrphostinsmay be useful in reducing or preventing the development of septic shockduring an inflammatory response. The tyrphostins may also be useful asanti-inflammatory agents and in counteracting pathogenic effectsmediated by various cytokines (c.g. TNF and IL-1) which occur forexample in autoimmunity.

1. Pharmaceutical compositions for countering damage to cells or tissuecomprising an effective amount of a compound of the general formula

wherein: Ar is a group of the formulae

n is 0 or, when Ar has the formula (i) above, then n may also be 1, R isCN, —C(S)NH₂, —C(O)NHR₃ or, when R₁ is 4-NO₂ and R₂ is H or 3-OH, then Rmay also be a group of the formula

where R₃ is H, phenyl, phenyl(lower alkyl) or pyridylmethyl; R₁ and R₂are each independently H, OH, NO₂ or, when R is CN, also CH₃, F, or CF₃,provided that both R₁ and R₂ arc not simultaneously H, together with apharmaceutically acceptable carrier.
 2. Pharmaceutical compositionsaccording to claim 1, comprising a compound of formula I wherein R isCN, —C(S)NH₂, —C(O)NHCH₂C₆H₅ or a group of the formula

and n is 0, R₁ is 4-NO₂ and R₂ is H.
 3. Pharmaceutical compositionsaccording to claim 1 or 2, for countering damage caused by a cytotoxicdrug.
 4. Pharmaceutical compositions according, to claim 3 forcountering damage caused by an anti-neoplastic drug.
 5. Pharmaceuticalcompositions according to claim 3 or 4 comprising, an effective amountof a compounds of formula I in claim 1, in combination with a cytotoxicdrug.
 6. Pharmaceutical compositions according to any one of claims 3-5,wherein the drug is selected from the group consisting of cisplatin,doxorubicin, cyclophosphamide, mitomycin-C and 5-fluorouracil.
 7. Apharmaceutical composition according to any one of claims 1-6, forcountering, myelotoxicity and lymphotoxicity.
 8. Pharmaceuticalcompositions according to any of the preceding claims in a form suitablefor intravenous administration.
 9. Pharmaceutical compositions accordingto any of the preceding claims in a form suitable for oraladministration.
 10. Pharmaceutical compositions according to claim 1 or2, for use in ex vivo preservation of cells, tissue or organ. 11.Pharmaceutical compositions according to claim 1 or 2, for counteringdamage caused by an immune mediated or inflammatory response.
 12. Apharmaceutical composition according to any one of claims 1-10, forcountering undesired apoptosis.
 13. A method of treatment of anindividual for countering damage to cells, tissue or organ, said methodcomprising administering to the individual an effective amount of acompound of the general formula

wherein: Ar is a group of the formulae

n is 0 or, when Ar has the formula (i) above, then n may also be 1, R isCN, —C(S)NH₂, —C(O)NHR₃ or, when R₁ is 4-NO₂ and R₂ is H or 3-OH, then Rmay also be a group of the formula

where R₃ is H, phenyl, phenyl(lower alkyl) or pyridylmethyl; R₁ and R₂arc each independently H, OH, NO₂ or, when R is CN, also CH₃, F, or CF₃,provided that both R₁ and R₂ are not simultaneously H, together with apharmaceutically acceptable carrier.
 14. A method according to claim 13,wherein in the compound of formula I, R is CN, —C(S)NH₂, —C(O)NHCH₂C₆H₅or a group of the formula

and n is 0, R₁ is 4-NO₂ and R₂ is H.
 15. A method according to claim 13or 14, wherein the harmful agent is a cytotoxic drug.
 16. A methodaccording to claim 15, wherein the anti-cytotoxic drug is ananti-neoplastic drug.
 17. A method according to claim 16, wherein thecytotoxic drug is selected from the group consisting of cisplatin,doxorubicin, cyclophosphamide, mitomycin-C and 5-fluorouracil.
 18. Amethod according to any one of claims 13-17, for counteringmyelotoxicity and lymphotoxicity.
 19. A method according to claim 12,for counting undesired harmful effects of irradiation treatment.
 20. Amethod according to any one of claims 13 to 19, wherein the compound offormula I is administered in combination with the cytotoxic drug orirradiation treatment.
 21. A method according to any one of claims 1 to20, wherein the compound of formula I is administered prior to theadministration of the cytotoxic drug or the irradiation treatment.
 22. Amethod according to any one of claims 13 to 21, wherein the compound offormula I is administered intravenously.
 23. A method according to anyone of claims 13 to 21, wherein the compounds of formula I isadministered orally.
 24. A method according to claim 13 or 14, forcountering damage caused by an immune mediated or inflammatory response.25. A method for ex vivo preservation of organ, tissue or cellscomprising contacting the organ, tissue or cells en vivo with a compoundof the general formula

wherein: Ar is a group of the formulae

n is 0 or, when Ar has the formula (i) above, then n may also be 1, R isCN, —C(S)NH₂, —C(O)NHR₃ or, when R₁ is 4-NO₂ and R₂ is H or 3-OH, then Rmay also be a group of the formula

where R₃ is H, phenyl, phenyl(lower alkyl) or pyridylmethyl; R₁ and R₂are each independently H, OH, NO₂ or, when R is CN, also CH₃, F, or CF₃,provided that both R₁ and R₂ are not simultaneously H, together with apharmaceutically acceptable carrier.
 26. Compound of the general formula

wherein Ar is a group of the formula

n is 0, or, when Ar has the formula (i) above, then n may also be 1, Ris —CN, —C(S)NH₂, —C(O)NHR₃ or, where R₁ is 4-NO₂ and R₂ is H, then Rmay also be a group of the formulae

R₁ and R₂ are each independently H, OH, NO₂ or, when R is CN, also CH₃,F or CF₃; and R₃ is H, phenyl, phenyl(lower alkyl) or pyridylmethyl;with the provisos that: a) when R is CN and n is 0, then (aa) if one ofR₁ and R₂ is H or OH, then the other cannot represent NO₂; (ab) if oneof R₁ and R₂ is H or F, then the other cannot represent H or F; and b)when R₁ is 4-NO₂, R₂ is H and n is 0, then R cannot represent —C(O)NH₂or —C(S)NH₂.
 27. Compound according to claim 26, wherein R is CN,—C(S)NH₂, —C(O)NHCH₂C₆H₅ or a group of the formula

and n is O,R₁ is 4-NO₂, R₂ is H and n is 0.